Microdissection Of The Chromosome And High-throughput Sequencing Of Chromosome 8 In Hevea Brasiliensis

Rubber is a kind of important strategic material in China.Hevea brasiliensis has become the only commercial source of natural rubber around the world for its high yield,excellent quality,ease to collect and long economic life.So it is of great significance to make the intensive study on rubber tree genome.At present,researchers had cloned more than 420 kinds of important genes or cDNA from Hevea brasiliensis.However,these genes were obtained from the rubber tree genome or cDNA library,which not only needs heavy work but also is unable to confirm their location and linkage relationships on chromosomes.Since the saturation about the genetic maps of rubber tree which constructed by a variety of molecular markers was not enough,it was difficult to be used for rubber tree breeding research.At present,related researches on the rubber tree libraries were only about the whole genome or cDNA libraries and the research on Hevea brasiliensis single chromosome library construction has not been reported.This study,started from the single chromosome of rubber tree,by the mean of Hevea brasiliensis single chromosome microdissection system which was established and perfected by our group members,and then carried out high-throughput sequencing,using a single-cell whole genome amplification method to do amplification instead of original LA-PCR amplification method,aimed to provide a new perspective for the study of rubber tree genome and lay a foundation for molecular assisted breeding of rubber tree.The results of the study are as follows:In this study,chromosome preparations of young leaves of Reyan 7-33-97 were made by using the enzyme and low osmotic method.The method of preparing chromosome samples was optimized which is suitable for Hevea brasiliensis single chromosome microdissection:early low osmosis for 40 min,enzymolysis for 7 h 20 min and later low osmosis for 30 min.Thirty-two chromosomes were successfully isolated from different metaphase cells of Reyan 7-33-97 by using the method of slender glass needle(Part of the chromosome has been separated repeatedly),and there were 7 chromosomes from one metaphase cell of Reyan 7-33-97.The single-celled whole genome amplification kit for in vitro amplification of single chromosome was optimized:The round of exponential amplification was optimized to 21 times instead of 17 times;the template quantity of the second round-exponential amplification was optimized to 2 μg,and the number of cycles was optimized to 10 times instead of 17 times.The effect of amplification product was better after optimizing.One of 32 separated chromosomes was selected to be amplified by single-cell whole genome amplification method.The product was verified by Southern blot hybridization.The product was homogeneous with the Hevea brasiliensis genomic DNA,indicating that DNA from the chromosome has been successfully amplified.The chromosome was verified by fluorescence in situ hybridization(FISH)and karyotype analysis and it was the chromosome 8 in Hevea brasiliensis.It showed that single-celled whole genome amplification kit can be used for in vitro amplification of Hevea brasiliensis single chromosome.We conducted a statistical analysis after high-throughput sequencing and data filtering,then we obtained 25479281 valid sequences which reads were 100 bp long and 5,262,483 of them were homogeneous with the sequences of Hevea brasiliensis,at a rate of 15.53%.Then after variation detecting,we obtained 112,616 SNP loci,724 InDel sites and 20 SV sites.These results can provide basic data for mutation pattern,the evolution of the species and the enriching saturation of genetic map.In this study,5000 sequences were selectedrandomly from clean reads after double filtration,and from among them,500 sequenceswere selected randomly to make comparisons with the WGS database,30 of them have more than 90%sequence homology with whole genome of rubber trees(RRIM600);and then 500 above-mentionedsequences were made comparisons with theEST database on NCBI,only 4 of them has high homology with ESTsequences.More valuable information requires further exploration.